Diagnosis of diseases associated with gene regulation

ABSTRACT

The present invention relates to the chemically modified genomic sequences of genes associated with gene regulation, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genes associated with gene regulation which are directed against the sequence, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes associated with gene regulation.

FIELD OF THE INVENTION

[0001] The levels of observation that have been well studied by themethodological developments of recent years in molecular biology, arethe genes themselves, the translation of these genes into RNA, and theresulting proteins. The question of which gene is switched on at whichpoint in the course of the development of an individual, and how theactivation and inhibition of specific genes in specific cells andtissues are controlled is correlatable to the degree and character ofthe methylation of the genes or of the genome. In this respect,pathogenic conditions may manifest themselves in a changed methylationpattern of individual genes or of the genome.

[0002] The present invention relates to nucleic acids, oligonucleotides,PNA-oligomers and to a method for the diagnosis and/or therapy ofdiseases which have a connection with the genetic and/or epigeneticparameters of genes associated with gene regulation and, in particular,with the methylation status thereof.

PRIOR ART

[0003] The completion of the sequencing of the human genome hashighlighted the need to further understand the regulation and control ofgenes. The cell exerts multiple levels of control over genes, howeverfor the majority of genes the most important regulatory mechanism istranscriptional control.

[0004] There are multiple mechanisms of gene regulation involvingdifferent families of proteins, genes and regulatory sequences. Thetranscription of eukaryotic genes by polymerases requires the priorassembly of transcription factors at the promoter region of the gene.These factors bind in a specific order, beginning with the binding ofTFIID to the TATA box. This provides several stages at which generegulation may take place, and many eukaryotic gene regulationmechanisms are thought to work by exerting positive or negative controlover these processes.

[0005] Gene regulatory proteins recognise short stretches of doublestranded DNA. Hundreds of such sequences have been identified throughoutthe genome, e.g. Spl, which recognises the sequence GGGCGG (in doublestranded form), GATA-1 which recognises the sequence TGATAG (in doublestranded form) and Oct-1 which recognises ATGCAAAT (in double strandedform). Gene regulatory proteins contain structural motifs that are ableto recognise the regulatory sequences using the unique pattern ofhydrogen bond donors, hydrogen bond acceptors and hydrophobic patches inthe major and minor groove of the DNA. Several famillies of structuralmotifs are known, including the helix-turn-helix motif, comprised ofamino acids, the zinc finger motif, which utilises one or more moleculesof zinc as a structural component, the leucine zipper andhelix-loop-helix motif.

[0006] Other mechanisms of gene regulation include chromatin packaging.Chromatin that is highly compacted is incapable of transcription, and ina less condensed form, DNA is still packaged on nucleosomes. Nucleosomespositioned at the beginning of a transcription site block the assemblyof transcription factors, and these must be removed. In addition genetranscription requires extensive winding and unwinding of thesupercoiled DNA. Although the exact mechanisms of DNA packaging are notcompletely understood, they include the use of a variety of DNAhelicases, gyrases and topoisomerases. Further discussion of generegulation may be found in standard molecular biology textbooks e.g.Alberts et. al. ‘Molecular Biology of the cell’ Garland Publishing.

[0007] Disruptions to gene regulation pathways have been implicated in awide variety of diseases including, for example, but not limited to:

[0008] Severe combined inununodeficiency disease:Misaki et. al.‘Gene-Transferred Oligoclonal T Cells Predominantly Persist inPeripheral Blood from an Adenosine Deaminase-Deficient Patient duringGene Therapy.’ Mol Ther 2001 Jan;3(l):24-27.

[0009] Cardiac damage: Pieper et. al. ‘Myocardial postischemic injury isreduced by polyADPri-pose polymerase-1 gene disruption.’ Mol Med 2000Apr;6(4):271-82.

[0010] Inflammatory response: Lekstrom-Himes J, Xanthopoulos KG‘CCAAT/enhancer binding protein epsilon is critical for effectiveneutrophil-mediated response to inflammatory challenge.’ Blood 1999 May1;93(9):3096-105.

[0011] Haemophilia B: Crossley M, Brownlee GG ‘Disruption of a C/EBPbinding site in the factor IX promoter is associated with haemophiliaB.’ Nature 1990 May 31;345(6274):444-6.

[0012] Werner syndrome: Li B, Comai L ‘Requirements for the nucleoyticprocessing of DNA ends by the Werner syndrome protein:Ku70/80 complex.’J Biol Chem Jan. 4, 2001.

[0013] Asthma: Nakamura et. al. ‘Gene expression of the GATA-3transcription factor is increased in atopic asthma.’ J Allergy ClinImmunol February 1999;103(2 Pt 1):215-22.

[0014] HDR syndrome: Van Esch et. al. ‘GATA3 haplo-insufficiency causeshuman HDR syndrome.’ Nature Jul. 27, 2000 ;406(6794):419-22.

[0015] Congenital heart defects: Srivastava D ‘HAND proteins: molecularmediators of cardiac development and congenital heart disease.’ TrendsCardiovasc Med January-February 1999;9(1-2):11-8.

[0016] Saethre-Chotzen syndrome: El Ghouzzi et. al. ‘Mutations within orupstream of the basic helix-loop-helix domain of the TWIST gene arespecific to Saethre-Chotzen syndrome.’ Eur J Hum Genet January1999;7(1):27-33.

[0017] Renal disease: Morello et. al. ‘Regulation of glomerular basementmembrane collagen expression by LMX1B contributes to renal disease innail patella syndrome.’ Nat Genet. Febuary 2001;27(2):205-208.

[0018] Preeclampsia: Rajakumar et. al. ‘Selective Overexpression of theHypoxia-Inducible Transcription Factor, HIF-2alpha, in Placentas fromWomen with Preeclampsia.’ Biol Reprod. February 2001;64(2):499-506.

[0019] Ogata et. al. ‘Inducible expression of basic transcriptionfactor-binding protein 2 (BTEB2), a member of zinc finger family oftranscription factors, in cardiac allograft vascular disease.’Transplantation. Dec. 15, 2000;70(11):1653-6.

[0020] Colorectal cancer: Rask et. al. ‘Increased expression of thetranscription factors CCAAT-enhancer binding protein-beta (C/EBBeta) andC/EBzeta (CHOP)correlate with invasive-ness of human colorectal cancer.’Int J Cancer. May 1, 2000;86(3):337-43.

[0021] Thyroid cancer: Kebebew et. al. ‘The helix-loop-helixtranscription factor, Id-1, is over-expressed in medullary thyroidcancer’ Surgery. December 2000;128(6):952-7.

[0022] Esophagal cancer: Saeki et. al. ‘Expression of ets-1transcription factor is correlated with penetrating tumor progression inpatients with squamous cell carcinoma of the esophagus’ Cancer. Oct. 15,2000;89(8):1670-6.

[0023] The diversity of mechanisms involved in the regulation of genesprovides an alternative level at which to target therapies and diagnosisfor diseases. In particular this may be relevant to diseases wherecurrent therapies may have unwanted side effects or fail to provideeffective treatment. For cancer patients such methods constitute aconsiderable advantage over conventional methods such as chemotherapywith their massive side effects, which sometimes result in unacceptablemorbidity or lead up to the death of the patient. In practice, theseunwanted side effects associated with cancer therapies frequently limitthe treatment which could help a patient.

[0024] A global analysis of the status of gene regulatory mechanismswould provide a basis for the development of appropriate and specifictherapies for the many diseases associated with gene regulation. Suchanalysis may be carried out in a gene specific manner based on theresults of gene expression, e.g. DNA micro array analysis of MRNAexpression or proteomic analysis. The next step would then be to look atthe causal factors involved at earlier stages in the regulatorymechanisrns. DNA methylation provides a novel level of information atwhich to analyse the genome.

[0025] 5-methylcytosine is the most frequent covalent base modificationin the DNA of eukaryotic cells. It plays a role, for example, in theregulation of the transcription, in genetic imprinting, and intumorigenesis. Therefore, the identification of 5-methylcytosine as acomponent of genetic information is of considerable interest. However,5-methylcytosine positions cannot be identified by sequencing since5-methylcytosine has the same base pairing behavior as cytosine.Moreover, the epigenetic information carried by 5-methylcytosine iscompletely lost during PCR amplification.

[0026] A relatively new and currently the most frequently used methodfor analyzing DNA for 5-methylcytosine is based upon the specificreaction of bisulfite with cytosine which, upon subsequent alkalinehydrolysis, is converted to uracil which corresponds to thymidine in itsbase pairing behavior. However, 5-methylcytosine remains unmodifiedunder these conditions. Consequently, the original DNA is converted insuch a manner that methylcytosine, which originally could not bedistinguished from cytosine by its hybridization behavior, can now bedetected as the only remaining cytosine using “normal” molecularbiological techniques, for example, by amplification and hybridizationor sequencing. All of these techniques are based on base pairing whichcan now be fully exploited. In terms of sensitivity, the prior art isdefined by a method which encloses the DNA to be analyzed in an agarosematrix, thus preventing the diffusion and renaturation of the DNA(bisulfite only reacts with single-stranded DNA), and which replaces allprecipitation and purification steps with fast dialysis (Olek A, OswaldJ, Walter J. A modified and improved method for bisulphite basedcytosine methylation analysis. Nucleic Acids Res. Dec. 15,1996;24(24):5064-6). Using this method, it is possible to analyzeindividual cells, which illustrates the potential of the method.However, currently only individual regions of a length of up toapproximately 3000 base pairs are analyzed, a global analysis of cellsfor thousands of possible methylation events is not possible. However,this method cannot reliably analyze very small fragments from smallsample quantities either. These are lost through the matrix in spite ofthe diffusion protection.

[0027] An overview of the further known methods of detecting5-methylcytosine may be gathered from the following review article:Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998,26,2255.

[0028] To date, barring few exceptions (e.g., Zeschnigk M, Lich C,Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for thediagnosis of Angelman and Prader-Willi syndrome based on allelicmethylation differences at the SNRPN locus. Eur J Hum Genet. March-April1997;5(2):94-8) the bisulfite technique is only used in research.Always, however, short, specific fragments of a known gene are amplifiedsubsequent to a bisulfite treatment and either completely sequenced(Olek A, Walter J. The pre-implantation ontogeny of the H19 methylationimprint. Nat Genet. November 1997;17(3):275-6) or individual cytosinepositions are detected by a primer extension reaction (Gonzalgo ML,Jones PA. Rapid quantitation of methylation differences at specificsites using methylation-sensitive single nucleotide primer extension(Ms-SNuPE). Nucleic Acids Res. Jun. 15, 1997;25(12):2529-31, Patentapplication WO 95/00669) or by enzymatic digestion (Xiong Z, Laird PW.COBRA: a sensitive and quantitative DNA methylation assay. Nucleic AcidsRes. Jun, 15 1997;25(12):2532-4). In addition, detection byhybridization has also been described (Olek et al., WO 99/28498).

[0029] Further publications dealing with the use of the bisulfitetechnique for methylation detection in individual genes are: Grigg G,Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays.June 1994;16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, BuitingK, Horsthemke B, Doerfler W. Imprinted segments in the human genome:different DNA methylation patterns in the Prader-Willi/Angelman syndromeregion as determined by the genomic sequencing method. Hum Mol Genet.March 1997;6(3):387-95; Feil R, Charlton J, Bird AP, Walter J, Reik W.Methylation analysis on individual chromosomes: improved protocol forbisulphite genomic sequencing. Nucleic Acids Res. Feb. 25,1994;22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio MC, Dante R.Genomic sequencing indicates a correlation between DNA hypomethylationin the 5′ region of the pS2 gene and its expression in human breastcancer cell lines. Gene. May 19, 1995;157(1-2):261-4; WO 97/46705, WO95/15373 and WO 97/45560.

[0030] An overview of the Prior Art in oligomer array manufacturing canbe gathered from a special edition of Nature Genetics (Nature GeneticsSupplement, Volume 21, January 1999), published in January 1999, andfrom the literature cited therein.

[0031] Fluorescently labeled probes are often used for the scanning ofimmobilized DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the5′-OH of the specific probe are particularly suitable for fluorescencelabels. The detection of the fluorescence of the hybridized probes maybe carried out, for example via a confocal microscope. Cy3 and Cy5 dyes,besides many others, are commercially available.

[0032] Matrix Assisted Laser Desorption Ionization Mass Spectrometry(MALDI-TOF) is a very efficient development for the analysis ofbiomolecules (Karas M, Hillenkamp F. Laser desorption ionization ofproteins with molecular masses exceeding 10,000 daltons. Anal Chem. Oct.15, 1988;60(20):2299-301). An analyte is embedded in a light-absorbingmatrix. The matrix is evaporated by a short laser pulse thustransporting the analyte molecule into the vapor phase in anunfragmented manner. The analyte is ionized by collisions with matrixmolecules. An applied voltage accelerates the ions into a field-freeflight tube. Due to their different masses, the ions are accelerated atdifferent rates. Smaller ions reach the detector sooner than biggerones.

[0033] MALDI-TOF spectrometry is excellently suited to the analysis ofpeptides and proteins. The analysis of nucleic acids is somewhat moredifficult (Gut I G, Beck S. DNA and Matrix Assisted Laser DesorptionIonization Mass Spectrometry. Current Innovations and Future Trends.1995, 1; 147-57). The sensitivity to nucleic acids is approximately 100times worse than to peptides and decreases disproportionally withincreasing fragment size. For nucleic acids having a multiply negativelycharged backbone, the ionization process via the matrix is considerablyless efficient. In MALDI-TOF spectrometry, the selection of the matrixplays an eminently important role. For the desorption of peptides,several very efficient matrixes have been found which produce a very fmecrystallization. There are now several responsive matrixes for DNA,however, the difference in sensitivity has not been reduced. Thedifference in sensitivity can be reduced by chemically modifying the DNAin such a manner that it becomes more similar to a peptide.Phosphorothioate nucleic acids in which the usual phosphates of thebackbone are substituted with thiophosphates can be converted into acharge-neutral DNA using simple alkylation chemistry (Gut IG, Beck S. Aprocedure for selective DNA alkylation and detection by massspectrometry. Nucleic Acids Res. Apr. 25, 1995 ;23(8):1367-73). Thecoupling of a charge tag to this modified DNA results in an increase insensitivity to the same level as that found for peptides. A furtheradvantage of charge tagging is the increased stability of the analysisagainst impurities which make the detection of unmodified substratesconsiderably more difficult.

[0034] Genomic DNA is obtained from DNA of cell, tissue or other testsamples using standard methods. This standard methodology is found inreferences such as Fritsch and Maniatis eds., Molecular Cloning: ALaboratory Manual, 1989.

DESCRIPTION

[0035] The object of the present invention is to provide the chemicallymodified DNA of genes associated with gene regulation, as well asoligonucleotides and/or PNA-oligomers for detecting cytosinemethylations, as well as a method which is particularly suitable for thediagnosis and/or therapy of genetic and epigenetic parameters of genesassociated with gene regulation. The present invention is based on thediscovery that genetic and epigenetic parameters and, in particular, thecytosine methylation pattern of genes associated with gene regulationare particularly suitable for the diagnosis and/or therapy of diseasesassociated with gene regulation.

[0036] This objective is achieved according to the present inventionusing a nucleic acid containing a sequence of at least 18 bases inlength of the chemically pretreated DNA of genes associated with generegulation according to one of Seq. ID No.1 through Seq. ID No.224 andsequences complementary thereto and/or a sequence of a chemicallypretreated DNA of genes according to table 1 and sequences complementarythereto. In the table, after the listed gene designations, therespective data bank numbers (accession numbers) are specified whichdefine the appertaining gene sequences as unique. GenBank was used asthe underlying data bank, which is located at the National Institute ofHealth, internet address www.ncbi.nlm.nih.gov.

[0037] The chemically modified nucleic acid could heretofore not beconnected with the ascertainment of genetic and epigenetic parameters.

[0038] The object of the present invention is further achieved by anoligonucleotide or oligomer for detecting the cytosine methylation statein chemically pretreated DNA, containing at least one base sequencehaving a length of at least 13 nucleotides which hybridizes to achemically pretreated DNA of genes associated with generegulationaccording to Seq. ID No.1 through Seq. ID No.224 and sequencescomplementary thereto and/or a sequence of a chemically pre-treated DNAof genes according to table 1 and sequences complementary thereto. Theoligomer probes according to the present invention constitute importantand effective tools which, for the first time, make it possible toascertain the genetic and epigenetic parameters of genes associated withgene regulation. The base sequence of the oligomers preferably containsat least one CpG dinucleotide. The probes may also exist in the form ofa PNA (peptide nucleic acid) which has particularly preferred pairingproperties. Particularly preferred are oligonucleotides according to thepresent invention in which the cytosine of the CpG dinucleotide is the5^(th)-9^(th) nucleotide from the 5′-end of the 13-mer; in the case ofPNA-oligomers, it is preferred for the cytosine of the CpG dinucleotideto be the 4^(th)-6^(th) nucleotide from the 5′-end of the 9-mer.

[0039] The oligomers according to the present invention are normallyused in so called “sets” which contain at least one oligomer for each ofthe CpG dinucleotides of the sequences of Seq. ID No.1 through Seq. IDNo.224 and sequences complementary thereto and/or a sequence of achemically pretreated DNA of genes according to table 1 and sequencescomplementary thereto. Preferred is a set which contains at least oneoligomer for each of the CpG dinucleotides from one of Seq. ID No.1through Seq. ID No.224 and sequences complementary thereto and/or asequence of a chemically pretreated DNA of genes according to table Iand sequences complementary thereto.

[0040] Moreover, the present invention makes available a set of at leasttwo oligonucleotides which can be used as so-called “primeroligonucleotides” for amplifying DNA sequences of one of Seq. ID No.1through Seq. ID No.224 and sequences complementary thereto and/or asequence of a chemically pretreated DNA of genes according to table 1and sequences complementary thereto, or segments thereof.

[0041] In the case of the sets of oligonucleotides according to thepresent invention, it is preferred that at least one oligonucleotide isbound to a solid phase.

[0042] The present invention moreover relates to a set of at least 10 n(oligonucleotides and/or PNA-oligomers) used for detecting the cytosinemethylation state in chemically pretreated genomic DNA (Seq. ID No.1through Seq. ID No.224 and sequences complementary thereto and/or asequence of a chemically pretreated DNA of genes according to table 1and sequences complementary thereto). These probes enable diagnosisand/or therapy of genetic and epigenetic parameters of genes associatedwith gene regulation. The set of oligomers may also be used fordetecting single nucleotide polymorphisms (SNPs) in the chemicallypretreated DNA of genes associated with gene regulation according to oneof Seq. ID No.1 through Seq. ID No.224 and sequences complementarythereto and/or a sequence of a chemically pretreated DNA of genes toaccording to table I and sequences complementary thereto. According tothe present invention, it is preferred that an arrangement of differentoligonucleotides and/or PNA-oligomers (a so-called “array”) madeavailable by the present invention is present in a manner that it islikewise bound to a solid phase. This array of differentoligonucleotide- and/or PNA-oligomer sequences can be characterized inthat it is arranged on the solid phase in the form of a rectangular orhexagonal lattice. The solid phase surface is preferably composed ofsilicon, glass, polystyrene, aluminum, steel, iron, copper, nickel,silver, or gold. However, nitrocellulose as well as plastics such asnylon which can exist in the form of pellets or also as resin matricesare possible as well.

[0043] Therefore, a further subject matter of the present invention is amethod for manufacturing an array fixed to a carrier material foranalysis in connection with diseases associated with gene regulationinwhich method at least one oligomer according to the present invention iscoupled to a solid phase. Methods for manufacturing such arrays areknown, for example, from U.S. Pat. No. 5,744,305 by means of solid-phasechemistry and photolabile protecting groups. A further subject matter ofthe present invention relates to a DNA chip for the analysis of diseasesassociated with gene regulationwhich contains at least one nucleic acidaccording to the present invention. DNA chips are known, for example,from U.S. Pat. No. 5,837,832.

[0044] Moreover, a subject matter of the present invention is a kitwhich may be composed, for example, of a bisulfite-containing reagent, aset of primer oligonucleotides containing at least two oligonucleotideswhose sequences in each case correspond or are complementary to an 18base long segment of the base sequences specified in the appendix (Seq.ID No.1 through Seq. ID No.224 and sequences complementary theretoand/or a sequence of a chemically pretreated DNA of genes according totable 1 and sequences complementary thereto), oligonucleotides and/orPNA-oligomers as well as instructions for carrying out and evaluatingthe described method. However, a kit along the lines of the presentinvention can also contain only part of the aforementioned components.

[0045] The present invention also makes available a method forascertaining genetic and/or epigenetic parameters of genes associatedwith the cycle cell by analyzing cytosine methylations and singlenucleotide polymorphisms, including the following steps:

[0046] In the first step of the method, a genomic DNA sample ischemically treated in such a manner that cytosine bases which areumnethylated at the 5′-position are converted to uracil, thyrnine, oranother base which is dissimilar to cytosine in terms of hybridizationbehavior. This will be understood as ‘chemical pretreatment’hereinafter.

[0047] The genomic DNA to be analyzed is preferably obtained form usualsources of DNA such as cells or cell components, for example, celllines, biopsies, blood, sputum, stool, urine, cerebral-spinal fluid,tissue embedded in paraffin such as tissue from eyes, intestine, kidney,brain, heart, prostate, lung, breast or liver, histologic object slides,or combinations thereof.

[0048] The above described treatment of genomic DNA is preferablycarried out with bisulfite (hydrogen sulfite, disulfite) and subsequentalkaline hydrolysis which results in a conversion of non-methylatedcytosine nucleobases to uracil or to another base which is dissimilar tocytosine in terms of base pairing behavior.

[0049] Fragments of the chemically pretreated DNA are amplified, usingsets of primer oligonucleotides according to the present invention, anda, preferably heat-stable polymerase. Because of statistical andpractical considerations, preferably more than ten different fragmentshaving a length of 100-2000 base pairs are amplified. The amplificationof several DNA segments can be carried out simultaneously in one and thesame reaction vessel. Usually, the amplification is carried out by meansof a polymerase chain reaction (PCR).

[0050] In a preferred embodiment of the method, the set of primeroligonucleotides includes at least two olignonucleotides whose sequencesare each reverse complementary or identical to an at least 18 base-pairlong segment of the base sequences specified in the appendix (Seq. IDNo.1 through Seq. ID No.224 and sequences complementary thereto and/or asequence of a chemically pretreated DNA of genes according to table 1and sequences complementary thereto). The primer oligonucleotides arepreferably characterized in that they do not contain any CpGdinucleotides.

[0051] According to the present invention, it is preferred that at leastone primer oligonucleotide is bonded to a solid phase duringamplification. The different oligonucleotide and/or PNA-oligomersequences can be arranged on a plane solid phase in the form of arectangular or hexagonal lattice, the solid phase surface preferablybeing composed of silicon, glass, polystyrene, aluminum, steel, iron,copper, nickel, silver, or gold, it being possible for other materialssuch as nitrocellulose or plastics to be used as well. The fragmentsobtained by means of the amplification can carry a directly orindirectly detectable label. Preferred are labels in the form offluorescence labels, radionuclides, or detachable molecule fragmentshaving a typical mass which can be detected in a mass spectrometer, itbeing preferred that the fragments that are produced have a singlepositive or negative net charge for better detectability in the massspectrometer. The detection may be carried out and visualized by meansof matrix assisted laser desorption/ionization mass spectrometry (MALDI)or using electron spray mass spectrometry (ESI).

[0052] The amplificates obtained in the second step of the method aresubsequently hybridized to an array or a set of oligonucleotides and/orPNA probes. In this context, the hybridization takes place in the mannerdescribed in the following. The set of probes used during thehybridization is preferably composed of at least 10 oligonucleotides orPNA-oligomers. In the process, the amplificates serve as probes whichhybridize to oligonucleotides previously bonded to a solid phase. Thenon-hybridized fragments are subsequently removed. Said oligonucleotidescontain at least one base sequence having a length of 13 nucleotideswhich is reverse complementary or identical to a segment of the basesequences specified in the appendix, the segment containing at least oneCpG dinucleotide. The cytosine of the CpG dinucleotide is the 5^(th) to9^(th) nucleotide from the 5′-end of the 13-mer. One oligonucleotideexists for each CpG dinucleotide. Said PNA-oligomers contain at leastone base sequence having a length of 9 nucleotides which is reversecomplementary or identical to a segment of the base sequences specifiedin the appendix, the segment containing at least one CpG dinucleotide.The cytosine of the CpG dinucleotide is the 4^(th) to 6^(th) nucleotideseen from the 5′-end of the 9-mer. One oligonucleotide exists for eachCpG dinucleotide.

[0053] In the fourth step of the method, the non-hybridized amplificatesare removed.

[0054] In the final step of the method, the hybridized amplificates aredetected. In this context, it is preferred that labels attached to theamplificates are identifiable at each position of the solid phase atwhich an oligonucleotide sequence is located.

[0055] According to the present invention, it is preferred that thelabels of the amplificates are fluorescence labels, radionuclides, ordetachable molecule fragments having a typical mass which can bedetected in a mass spectrometer. The mass spectrometer is preferred forthe detection of the amplificates, fragments of the amplificates or ofprobes which are complementary to the amplificates, it being possiblefor the detection to be carried out and visualized by means of matrixassisted laser desorption/ionization mass spectrometry (MALDI) or usingelectron spray mass spectrometry (ESI).

[0056] The produced fragments may have a single positive or negative netcharge for better detectability in the mass spectrometer. Theaforementioned method is preferably used for ascertaining genetic and/orepigenetic parameters of genes associated with gene regulation.

[0057] The oligomers according to the present invention or arraysthereof as well as a kit according to the present invention are intendedto be used for the diagnosis and/or therapy of diseases associated withgene regulationby analyzing methylation patterns of genes associatedwith gene regulation. According to the present invention, the method ispreferably used for the diagnosis and/or therapy of important geneticand/or epigenetic parameters within genes associated with generegulation.

[0058] The method according to the present invention is used, forexample, for the diagnosis and/or therapy of severe combinedimmunodeficiency disease, cardiac disorders, inflammatory response,heamophilia, Werner syndrome, asthma, HDR syndrome, Saethre-Chotzensyndrome, renal disease, preeclampsia, graft-vetsus-host disease, solidtumors and cancers.

[0059] The nucleic acids according to the present invention of Seq. IDNo.1 through Seq. ID No.224 and sequences complementary thereto and/or asequence of a chemically pretreated DNA of genes according to table 1and sequences complementary thereto can be used for the diagnosis and/ortherapy of genetic and/or epigenetic parameters of genes associated withgene regulation.

[0060] The present invention moreover relates to a method formanufacturing a diagnostic agent and/or therapeutic agent for thediagnosis and/or therapy of diseases associated with gene regulationbyanalyzing methylation patterns of genes associated with gene regulation,the diagnostic agent and/or therapeutic agent being characterized inthat at least one nucleic acid according to the present invention isused for manufacturing it, possibly together with suitable additives andauxiliary agents.

[0061] A further subject matter of the present invention relates to adiagnostic agent and/or therapeutic agent for diseases associated withgene regulationby analyzing methylation patterns of genes associatedwith gene regulation, the diagnostic agent and/or therapeutic agentcontaining at least one nucleic acid according to the present invention,possibly together with suitable additives and auxiliary agents.

[0062] The present invention moreover relates to the diagnosis and/orprognosis of events which are disadvantageous to patients or individualsin which important genetic and/or epigenetic parameters within genesassociated with gene regulation said parameters obtained by means of thepresent invention may be compared to another set of genetic and/orepigenetic parameters, the differences serving as the basis for adiagnosis and/or prognosis of events which are disadvantageous topatients or individuals.

[0063] In the context of the present invention the term “hybridization”is to be understood as a bond of an oligonucleotide to a completelycomplementary sequence along the lines of the Watson-Crick base pairingsin the sample DNA, forming a duplex structure. To be understood by“stringent hybridization conditions” are those conditions in which ahybridization is carried out at 60° C. in 2.5×SSC buffer, followed byseveral washing steps at 37° C. in a low buffer concentration, andremains stable.

[0064] The term “functional variants” denotes all DNA sequences whichare complementary to a DNA sequence, and which hybridize to thereference sequence under stringent conditions and have an activitysimilar to the corresponding polypeptide according to the presentinvention.

[0065] In the context of the present invention, “genetic parameters” aremutations and polymorphisms of genes associated with gene regulationandsequences further required for their regulation. To be designated asmutations are, in particular, insertions, deletions, point mutations,inversions and polymorphisms and, particularly preferred, SNPs (singlenucleotide polymorphisms).

[0066] In the context of the present invention, “epigenetic parameters”are, in particular, cytosine methylations and further chemicalmodifications of DNA bases of genes associated with gene regulationandsequences further required for their regulation. Further epigeneticparameters include, for example, the acetylation of histones which,however, cannot be directly analyzed using the described method butwhich, in turn, correlates with the DNA methylation. In the following,the present invention will be explained in greater detail on the basisof the sequences and examples with reference to the accompanying figurewithout being limited thereto.

[0067]FIG. 1

[0068]FIG. 1 shows the hybridisation of fluorescent labelledamplificates to a surface bound olignonucleotide. Sample I being frompilocytic astrocytoma (brain tunor) tissue and sample II being fromgrade II astrocytoma (brain tumor) tissue. Flourescence at a spot showshybridisation of the amplificate to the olignonucleotide. Hybridisationto a CG olignonucleotide denotes methylation at the cytosine positionbeing analysed, hybridisation to a TG olignonucleotide denotes nomethylation at the cytosine position being analysed.

[0069] Sequence ID Nos. 1 to 224

[0070] Sequence ID Nos. 1 to 224 show sequences of genes associated withgene regulation according to the invention. Sequences having oddsequence numbers (e.g., Seq. ID No. 1, 3, 5, . . .) exhibit in each casesequences of the chemically pretreated genomic DNAs of different genesassociated with gene regulation. Sequences having even sequence numbers(e.g., Seq. ID No. 2, 4, 6, . . . ) exhibit in each case the sequencesof the chemically pretreated genornic DNAs of genes associated with generegulationwhich are complementary to the preceeding sequences (e.g., thecomplementary sequence to Seq. ID No.1 is Seq. ID No.2, thecomplementary sequence to Seq. ID No.3 is Seq. ID No.4, etc.)

[0071] Sequence ID Nos. 225 to 228

[0072] Sequence ID Nos. 225 to 228 show the sequences ofoligonucleotides used in Example 1.

EXAMPLE 1

[0073] Methylation analysis of the gene p53 associated with generegulation.

[0074] This example relates to a fragment of a gene associated with generegulation, in this case, p53 in which a specific CG-position isanalyzed for its methylation status.

[0075] In the first step, a genomic sequence is treated using bisulfite(hydrogen sulfite, disulfite) in such a manner that all cytosines whichare not methylated at the 5-position of the base are modified in such amanner that a different base is substituted with regard to the basepairing behavior while the cytosines methylated at the 5-position remainunchanged.

[0076] If bisulfite solution is used for the reaction, then an additiontakes place at the non-methylated cytosine bases. Moreover, adenaturating reagent or solvent as well as a radical interceptor must bepresent. A subsequent alkaline hydrolysis then gives rise to theconversion of non-methylated cytosine nucleobases to uracil. Thechemically converted DNA (Seq ID No. 221) is then used for the detectionof methylated cytosines. In the second step of the method, the treatedDNA sample is diluted with water or an aqueous solution. Preferably, theDNA is subsequently desulfonated (10-30 min, 90-100° C.) at an alkalinepH value. In the third step of the method, the DNA sample is amplifiedin a polymerase chain reaction, preferably using a heat-resistant DNApolymerase. In the present case, cytosines of the gene p53 are analyzed.To this end, a defined fragment having a length of 595 bp is amplifiedwith the specific primer oligo- nucleotides GTGATAAGGGTTGTGAAGGA(sequence ID No.225) and CAAAAACTTACCCAATCCAA (sequence ID No.226). Thisamplificate serves as a sample which hybridizes to an oligonucleotidepreviously bonded to a solid phase, forming a duplex structure, forexample AACCCCTACGAAACTCCT (sequence ID No.227), the cytosine to bedetected being located at position 401 of the amplificate. The detectionof the hybridization product is based on Cy3 and Cy5 flourescentlylabeled primer oligonucleotides which have been used for theamplification. A hybridization reaction of the amplified DNA with theoligonucleotide takes place only if a methylated cytosine was present atthis location in the bisulfite-treated DNA. Thus, the methylation statusof the specific cytosine to be analyzed is inferred from thehybridization product.

[0077] In order to verify the methylation status of the position, asample of the amplificate is further hybridized to anotheroligonucleotide previously bonded to a solid phase. Saidolignonucleotide is identical to the oligonucleotide previously used toanalyze the methylation status of the sample, with the exception of theposition in question. At the position to be analysed saidoligonucleotide comprises a thymine base as opposed to a cytosine basei.e. sequence AACCCCTACAAAACTCCT (sequence ID No.228). Therefore, thehybridization reaction only takes place if an unmethylated cytosine waspresent at the position to be analysed.

[0078] The analysis was carried out on two tissue samples, Sample I frompilocytic astrocytoma (brain tumor) tissue, and Sample 2 from grade IIastrocytoma (brain tumor) tissue. From the results (FIG. 1) it can beseen that Sample 2 was methylated at position 401 of the amplificatewhereas Sample 1 contained a mixture of both methylated and unmethylatedcells at the same position.

EXAMPLE 2

[0079] Diagnosis of diseases associated with gene regulation

[0080] In order to relate the methylation patterns to one of thediseases associated with gene regulation, it is initially required toanalyze the DNA methylation patterns of a group of diseased and of agroup of healthy patients. These analyses are carried out, for example,analogously to example 1. The results obtained in this manner are storedin a database and the CpG dinucleotides which are methylated differentlybetween the two groups are identified. This can be carried out bydetermining individual CpG methylation rates as can be done, forexample, in a relatively imprecise manner, by sequencing or else, in avery precise manner, by a methylation-sensitive “primer extensionreaction”. It is also possible for the entire methylation status to beanalyzed simultaneously, and for the patterns to be compared, forexample, by clustering analyses which can be carried out, for example,by a computer.

[0081] Subsequently, it is possible to allocate the examined patients toa specific therapy group and to treat these patients selectively with anindividualized therapy.

[0082] Example 2 can be carried out, for example, for the followingdiseases: severe combined immunodeficiency disease, cardiac disorders,inflammatory response, heamophilia, Werner syndrome, asthma, HDRsyndrome, Saethre-Chotzen syndrome, renal disease, preeclampsia,graft-versus-host disease, solid tumors and cancers. TABLE 1 Listing ofpreferred genes associated with gene regulation according to theinvention. Database entry No. (GenBank, internet ad- Gene dresswww.ncbi.nlm.nih.gov) CBFB (L20298), ELF1 (M82882), ETV3 (L16464), ETV4(D12765), LYL1 (M22637), RENBP (U52112), TAF2C2 (Y09321), TCF3 (M31222),H4FI (NM_003544), ADA (NM_000022), ATF3 (NM_001674), CEBPD (NM_005195),CHD1L (NM_004284), CTPS (NM_001905), DCTD (NM_001921), EIF2B1(NM_001414), ELF3 (NM_004433), ELK4 (NM_021795), ETV5 (NM_004454),FOXO1A (NM_002015), HIVEP1 (NM_002114), HMG2 (NM_002129), ID1(NM_002165), ID3 (NM_002167), ID4 (NM_001546), LAF4 (NM_0022859), MAFG(NM_002359), MHC2TA (NM_000246), ODC1 (NM_002539), PBX3 (NM_006195),PCNA (NM_002592), POU2AF1 (NM_006235), PRPS1 (NM_002764), RARG(NM_000966), RECQL (NM_002907), RXRA (NM_002957), TIAL1 (NM_003252),ZNF173 (NM_003449), ATBF1 (NM_006885), FLI1 (NM_002017), NUMA1(NM_006185), POU2F2 (NM_002698), SF100 (NM_003113)

[0083]

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030082609). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A nucleic acid comprising a sequence at least 18 bases in length of asegment of the chemically pretreated DNA of genes associated with generegulation according to one of the sequences taken from the group ofSeq. ID No.1 to Seq. ID No.224 and sequences complementary thereto.
 2. Anucleic acid comprising a sequence at least 18 base pairs in length of asegment of the chemically pretreated DNA of genes associated with generegulation according to one of the sequences according to one of thegenes CBFB (L20298), ELF1 (M82882), ETV3 (L16464), ETV4 (12765), LYL1(M22637), RENBP (U52112), TAF2C2 (Y09321), TCF3 (M31222), H4FI(NM_(—)003544), ADA (NM_(—)000022), ATF3 (NM_(—)00674), CEBPD(NM_(—)005195), CHD1L (NM_(—)004284), CTPS (NM_(—)001905), DCTD(NM_(—)001921), EIF2B1 (NM_(—)001414), ELF3 (NM_(—)004433), ELK4(NM_(—)02795), ETV5 (NM_(—)004454), FOXOlA (NM_(—)002015), HIVEP1(NM_(—)002114), HMG2 (NM_(—)002129), ID1 (NM_(—)002165), ID3(NM_(—)002167), ID4 (NM_(—)001546), LAF4 (NM0022859), MAFG(NM_(—)002359), MHC2TA (NM_(—)000246), ODC1 (NM_(—)002539), pbx3(NM_(—)006195), pcna (NM_(—)002592), POU2AF1 (NM_(—)006235), prps1(NM_(—)002764), RARG (NM_(—)000966), recql (NM_(—)02907), RXRA(NM_(—)002957), TIAL1 (NM_(—)003252), ZNF173 (NM_(—)003449), ATBF1(NM_(—)006885), FLI1 (NM_(—)002017), NUMA1 (NM_(—)006185), POU2F2(NM_(—)002698), SP100(NM_(—)003113), and sequences complementarythereto.
 3. An oligomer, in particular an oligonucleotide or peptidenucleic acid (PNA)-oligomer, said oligomer comprising in each case atleast one base sequence having a length of at least 9 nucleotides whichhybridizes to or is identical to a chemically pretreated DNA of genesassociated with gene regulation according to one of the Seq ID Nos 1 to224 according to claim 1 or to a chemically pretreated DNA of genesaccording to claim 2 and sequences complementary thereto.
 4. Theoligomer as recited in claim 3; wherein the base sequence includes atleast one CpG dinucleotide
 5. The oligomer as recited in claim 3;characterized in that the cytosine of the CpG dinucleotide is locatedapproximately in the middle third of the oligomer.
 6. A set ofoligomers, comprising at least two oligomers according to any of claims3 to
 5. 7. A set of oligomers as recited in claim 6, comprisingoligomers for detecting the methylation state of all CpG dinucleotideswithin one of the sequences according to Seq. ID Nos. 1 through Seq. ID224 according to claim 1 or a chemically pretreated DNA of genesaccording to claim 2, and sequences complementary thereto.
 8. A set ofat least two oligonucleotides as recited in claim 3, which can be usedas primer oligonucleotides for the amplification of DNA sequences of oneof Seq. ID No. 1 through Seq. ID No.224 and sequences complementarythereto and/or sequences of a chemically pretreated DNA of genesaccording to claim 2, and sequences complementary thereto and segmentsthereof.
 9. A set of oligonucleotides as recited in claim 8,characterized in that at least one oligonucleotide is bound to a solidphase.
 10. Use of a set of oligomer probes comprising at least ten ofthe oligomers according to any of claims 6 through 9 for detecting thecytosine methylation state and/or single nucleotide polymorphisms (SNPs)in a chemically pretreated genomic DNA according to claim 1 or achemically pretreated DNA of genes according to claim
 2. 11. A methodfor manufacturing an arrangement of different oligomers (array) fixed toa carrier material for analyzing diseases associated with themethylation state of the CpG dinucleotides of one of the Seq. ID No. 1through Seq. ID No.224 and sequences complementary thereto and/orchemically pretreated DNA of genes according to claim 2, wherein atleast one oligomer according to any of the claims 3 through 5 is coupledto a solid phase.
 12. An arrangement of different oligomers (array)obtainable according to claim
 11. 13. An array of differentoligonucleotide- and/or PNA-oligomer sequences as recited in claim 12,characterized in that these are arranged on a plane solid phase in theform of a rectangular or hexagonal lattice.
 14. The array as recited inany of the claims 12 or 13, characterized in that the solid phasesurface is composed of silicon, glass, polystyrene, aluminum, steel,iron, copper, nickel, silver, or gold.
 15. A DNA- and/or PNA-array foranalyzing diseases associated with the methylation state of genes,comprising at least one nucleic acid according to one of the preceedingclaims.
 16. A method for ascertaining genetic and/or epigeneticparameters for the diagnosis and/or therapy of existing diseases or thepredisposition to specific diseases by analyzing cytosine methylations,characterized in that the following steps are carried out: a) in agenomic DNA sample, cytosine bases which are unmethylated at the5-position are converted, by chemical treatment, to uracil or anotherbase which is dissimilar to cytosine in terms of hybridization behavior;b) fragments of the chemically pretreated genomic DNA are amplifiedusing sets of primer oligonucleotides according to claim 8 or 9 and apolymerase, the amplificates carrying a detectable label; c)Amplificates are hybridized to a set of oligonucleotides and/or PNAprobes according to the claims 6 and 7, or else to an array according toone of the claims 12 through 15; d) the hybridized amplificates aresubsequently detected.
 17. The method as recited in claim 16,characterized in that the chemical treatment is carried out by means ofa solution of a bisulfite, hydrogen sulfite or disulfite.
 18. The methodas recited in one of the claims 16 or 17, characterized in that morethan ten different fragments having a length of 100-2000 base pairs areamplified.
 19. The method as recited in one of the claims 16 through 18,characterized in that the amplification of several DNA segments iscarried out in one reaction vessel.
 20. The method as recited in one ofthe claims 16 through 19, characterized in that the polymerase is aheat-resistant DNA polymerase.
 21. The method as recited in claim 20,characterized in that the amplification is carried out by means of thepolymerase chain reaction (PCR).
 22. The method as recited in one of theclaims 16 through 21, characterized in that the labels of theamplificates are fluorescence labels.
 23. The method as recited in oneof the claims 16 through 21, characterized in that the labels of theamplificates are radionuclides.
 24. The method as recited in one of theclaims 16 through 21, characterized in that the labels of theamplificates are detachable molecule fragments having a typical masswhich are detected in a mass spectrometer.
 25. The method as recited inone of the claims 16 through 21, characterized in that the amplificatesor fragments of the amplificates are detected in the mass spectrometer.26. The method as recited in one of the claims 24 and/or 25,characterized in that the produced fragments have a single positive ornegative net charge for better detectability in the mass spectrometer27. The method as recited in one of the claims 24 through 26,characterized in that detection is carried out and visualized by meansof matrix assisted laser desorption/ionization mass spectrometry (MALDI)or using electron spray mass spectrometry (ESI).
 28. The method asrecited in one of the claims 16 through 27, characterized in that thegenomic DNA is obtained from cells or cellular components which containDNA, sources of DNA comprising, for example, cell lines, biopsies,blood, sputum, stool, urine, cerebral-spinal fluid, tissue embedded inparaffin such as tissue from eyes, intestine, kidney, brain, heart,prostate, lung, breast or liver, histologic object slides, and allpossible combinations thereof.
 29. A kit comprising a bisulfite(=disulfite, hydrogen sulfite) reagent as well as oligonucleotidesand/or PNA-oligomers according to one of the claims 3 through
 5. 30. Theuse of a nucleic acid according to claims 1 or 2, of an oligonucleotideor PNA-oligomer according to one of the claims 3 through 5, of a kitaccording to claim 29, of an array according to one of the claims 12through 15, of a set of oligonucleotides according to one of claims 6through 9 for the diagnosis of severe combined immunodeficiency disease,cardiac disorders, inflammatory response, heamophilia, Werner syndrome,asthma, HDR syndrome, Saethre-Chotzen syndrome, renal disease,preeclampsia, graft-versus-host disease, solid tumors and cancers. 31.The use of a nucleic acid according to claims 1 or 2, of anoligonucleotide or PNA-oligomer according to one of claims 3 through 5,of a kit according to claim 29, of an array according to one of theclaims 12 through 15, of a set of oligonucleotides according to one ofcalims 6 through 9 for the therapy of severe combined immunodeficiencydisease, cardiac disorders, inflammatory response, heamophilia, Wernersyndrome, asthma, HDR syndrome, Saethre-Chotzen syndrome, renal disease,preeclampsia, graft-versus-host disease, solid tumors and cancers.
 32. Akit, comprising a bisulfite (=disulfite, hydrogen sulfite) reagent aswell as oligonucleotides and/or PNA-oligomers according to one of claims3 through 5.